LOCUS       sequence-324331-        6193 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6193)
  AUTHORS   Shen H, Forgacs D, Chapla D, Moremen KW, Wells L, Hamer SA, Tompkins
            SM, Ross TM, Rouphael N, Edupuganti S, Collins MH, Tarleton RL
  TITLE     A flexible, pan-species, multi-antigen platform for the detection 
            and monitoring of SARS-CoV-2-specific antibody responses.
  JOURNAL   medRxiv [Preprint]. 2021 Jan 27:2021.01.20.21249279. doi: 
            10.1101/2021.01.20.21249279.
  PUBMED    33532799
REFERENCE   2  (bases 1 to 6193)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "medRxiv 
            [Preprint]. 2021 Jan 27:2021.01.20.21249279. doi: 
            10.1101/2021.01.20.21249279."
COMMENT     Sequence Label: pET32a-Spike-403-505
FEATURES             Location/Qualifiers
     source          1..6193
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     647..666
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(686..763)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     969..988
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1076..1095
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1076..1094
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1095..1119
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1134..1156
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1164..1490
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             1512..1529
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1539..1556
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1563..1607
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1623..1637
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             1992..2009
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2062..2080)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2076..2123
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2160..2615
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2247..2266)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2457..2478
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2642..2746
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2747..2815,2816..3607)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2965..2984)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3778..4366
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4267..4286
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4520..4537
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4552..4694
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4620..4639)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4780..4802
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4796..4987)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5796..6193,1..685))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
       61 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
      121 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
      181 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
      241 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
      301 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
      361 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
      421 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
      481 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
      541 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
      601 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
      661 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
      721 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
      781 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
      841 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
      901 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
      961 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     1021 ccggccacga tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata
     1081 cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt
     1141 aactttaaga aggagatata catatgagcg ataaaattat tcacctgact gacgacagtt
     1201 ttgacacgga tgtactcaaa gcggacgggg cgatcctcgt cgatttctgg gcagagtggt
     1261 gcggtccgtg caaaatgatc gccccgattc tggatgaaat cgctgacgaa tatcagggca
     1321 aactgaccgt tgcaaaactg aacatcgatc aaaaccctgg cactgcgccg aaatatggca
     1381 tccgtggtat cccgactctg ctgctgttca aaaacggtga agtggcggca accaaagtgg
     1441 gtgcactgtc taaaggtcag ttgaaagagt tcctcgacgc taacctggcc ggttctggtt
     1501 ctggccatat gcaccatcat catcatcatt cttctggtct ggtgccacgc ggttctggta
     1561 tgaaagaaac cgctgctgct aaattcgaac gccagcacat ggacagccca gatctgggta
     1621 ccgacgacga cgacaaggcc atggctgata tcggatccat gggagatgaa gtaaggcaaa
     1681 tagctcccgg ccagaccggt aaaatcgctg attataacta caagctgccg gatgacttta
     1741 ccggctgcgt gattgcgtgg aattccaaca acttggacag caaggtgggc ggtaattaca
     1801 attacctgta ccgcctgttt cgtaaatcta acctgaagcc gtttgagcgt gacatctcga
     1861 ccgaaattta tcaggcaggt agcactccgt gtaatggcgt tgagggcttc aactgctact
     1921 tcccgttgca aagctatggt ttccagccaa cgaacggtgt tggttatcaa aagcttgcgg
     1981 ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     2041 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     2101 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     2161 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2221 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2281 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2341 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2401 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2461 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2521 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2581 cgcgaatttt aacaaaatat taacgtttac aatttcaggt ggcacttttc ggggaaatgt
     2641 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     2701 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     2761 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2821 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     2881 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     2941 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     3001 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3061 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3121 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3181 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3241 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     3301 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3361 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3421 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3481 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3541 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3601 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3661 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     3721 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     3781 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     3841 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     3901 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
     3961 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     4021 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     4081 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     4141 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     4201 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     4261 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     4321 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     4381 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     4441 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     4501 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     4561 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt
     4621 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     4681 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4741 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4801 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     4861 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     4921 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4981 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     5041 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     5101 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     5161 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     5221 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     5281 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     5341 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     5401 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     5461 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     5521 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     5581 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     5641 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     5701 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     5761 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5821 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5881 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     5941 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     6001 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     6061 tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     6121 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     6181 agcatttgca tgg
//
